Mechanisms of opsin activation.
Rhodopsin is constrained in an inactive conformation by interactions with 11-cis-retinal including formation of a protonated Schiff base with Lys296. Upon photoisomerization, major structural rearrangements that involve protonation of the active site Glu113 and cytoplasmic acidic residues, including Glu134, lead to the formation of the active form of the receptor, metarhodopsin II b, which decays to opsin. However, an activated receptor may be generated without illumination by addition of all-trans-retinal or its analogues to opsin, as measured in this study by the increased phosphorylation of opsin by rhodopsin kinase. The potency of stimulation depended on the chemical and isomeric nature of the analogues and the length of the polyene chain with all-trans-C17 aldehyde and all-trans-retinal being the most active and trans-C12 aldehyde being the least active. Certain cis-isomers, 11-cis-13-demethyl-retinal and 9-cis-C17 aldehyde, were also active. Most of the retinal analogues tested did not regenerate a spectrally identifiable pigment, and many were incapable of Schiff base formation (ketone, stable oximes, and Schiff base-derivatives of retinal). Thus, receptor activation resulted from formation of non-covalent complexes with opsin. pH titrations suggested that an equilibrium exists between partially active (protonated) and inactive (deprotonated) forms of opsin. These findings are consistent with a model in which protonation of one or more cytoplasmic carboxyl groups of opsin is essential for activity. Upon addition of retinoids, the partially active conformation of opsin is converted to a more active intermediate similar to metarhodopsin II b. The model provides an understanding of the structural requirements for opsin activation and an interpretation of the observed activities of natural and experimental opsin mutants.